Expression profiles of lncRNAs, miRNAs, and mRNAs and interaction analysis indicate their potential involvement during testicular fusion in Spodoptera litura.

Testicular fusion of Spodoptera litura occures during metamorphosis, which benefits sperms development. Previous research identified involvement of ECM-integrin interaction pathways, MMPs in testicular fusion, but the regulatory mechanism remains unclear. RNA-seq was performed to analyze long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in testes, aiming to uncover potential regulatory mechanisms of testicular fusion. 2150 lncRNAs, 2742 targeted mRNAs, and 347 miRNAs were identified in testes at three different developmental stages. Up-regulated DElncRNAs and DEmRNAs, as well as down-regulated DEmiRNAs, were observed during testicular fusion, while the opposite expression pattern was observed after fusion. Enrichment analysis of DEmRNAs revealed that cAMP signal pathway, ECM remodeling enzymes, ECM-integrin interaction pathways, and cell adhesion molecules were potentially associated with testicular fusion. The identified DElncRNA-DEmiRNA-DEmRNA regulatory network related to cAMP signal pathway, ECM remodeling enzymes suggests their roles during testicular fusion. Our research will provide new targets for studying the mechanism of testicular fusion.

DNA G-quadruplex structure participates in regulation of lipid metabolism through acyl-CoA binding protein.

G-quadruplex structure (G4) is a type of DNA secondary structure that widely exists in the genomes of many organisms. G4s are believed to participate in multiple biological processes. Acyl-CoA binding protein (ACBP), a ubiquitously expressed and highly conserved protein in eukaryotic cells, plays important roles in lipid metabolism by transporting and protecting acyl-CoA esters. Here, we report the functional identification of a G4 in the promoter of the ACBP gene in silkworm and human cancer cells. We found that G4 exists as a conserved element in the promoters of ACBP genes in invertebrates and vertebrates. The BmACBP G4 bound with G4-binding protein LARK regulated BmACBP transcription, which was blocked by the G4 stabilizer pyridostatin (PDS) and G4 antisense oligonucleotides. PDS treatment with fifth instar silkworm larvae decreased the BmACBP expression and triacylglycerides (TAG) level, resulting in reductions in fat body mass, body size and weight and growth and metamorphic rates. PDS treatment and knocking out of the HsACBP G4 in human hepatic adenocarcinoma HepG2 cells inhibited the expression of HsACBP and decreased the TAG level and cell proliferation. Altogether, our findings suggest that G4 of the ACBP genes is involved in regulation of lipid metabolism processes in invertebrates and vertebrates.

Juvenile hormone signaling promotes ovulation and maintains egg shape by inducing expression of extracellular matrix genes.

It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. Here, we describe a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce: the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JH-regulated insect reproduction.

Gut dysbacteriosis induces expression differences in the adult head transcriptome of Spodoptera frugiperda in a sex-specific manner.

Mounting evidence indicates that the gut microbiota influences the neurodevelopment and behavior of insects through the gut-brain axis. However, it is currently unclear whether the gut microbiota affect the head profiles and immune pathway in pests. Here, we find that gut bacteria is essential for the immune and neural development of adult Spodoptera frugiperda, which is an extremely destructive agricultural pest worldwide. 16 S rRNA sequencing analysis showed that antibiotics exposure significantly disturbed the composition and diversity of gut bacteria. Further transcriptomic analysis revealed that the adult head transcripts were greatly affected by gut dysbacteriosis, and differently expression genes critical for brain and neural development including A4galt, Tret1, nsun4, Galt, Mitofilin, SLC2A3, snk, GABRB3, Oamb and SLC6A1 were substantially repressed. Interestingly, the dysbacteriosis caused sex-specific differences in immune response. The mRNA levels of pll (serine/threonine protein kinase Pelle), PGRP (peptidoglycan-sensing receptor), CECA (cecropin A) and CECB (cecropin B) involved in Toll and Imd signaling pathway were drastically decreased in treated male adults' heads but not in female adults; however, genes of HIVEP2, ZNF131, inducible zinc finger protein 1-like and zinc finger protein 99-like encoding zinc-finger antiviral protein (ZAP) involved in the interferon (IFNα/ß) pathway were significantly inhibited in treated female adults' heads. Collectively, these results demonstrate that gut microbiota may regulate head transcription and impact the S. frugiperda adults' heads through the immune pathway in a sex-specific manner. Our finding highlights the relationship between the gut microbiota and head immune systems of S. frugiperda adults, which is an astonishing similarity with the discoveries of other animals. Therefore, this is the basis for further research to understand the interactions between hosts and microorganisms via the gut-brain axis in S. frugiperda and other insects.

Large-scale screening of i-motif binding compounds in the silkworm, Bombyx mori.

DNA secondary structure i-motif involves in gene transcription and considered as a novel target for cancer gene therapy. I-motif-binding compounds can either stabilize or destroy the structure, resulting in change in target gene transcription. In this study, a large-scale screening of binding compounds was conducted using the i-motif structure of BmPOUM2, a transcription factor in silkworm, Bombyx mori. Surface plasmon resonance imaging (SPRi) high-throughput binding screening of 3642 compounds found 60 compounds with an binding affinity Kd of 10-7-10-6 M. SPRi and circular dichroism (CD) double screening demonstrated that the BmPOUM2 i-motif structure bound the compounds IF1, IF3, IF4, IF6 and IF7 with Kd of 10-7 M, and the compounds IF2 and tetrakis (4-N-methylpyridyl) porphine (TMPyP4) with a Kd of 10-8 M. Interestingly, IF2, IF3, IF4, IF6 and IF7 promoted the binding of the i-motif-binding protein BmILF with the i-motif structure, whereas TMPyP4 inhibited the binding. This study provided a list of compounds that have potential applications in functional analysis of i-motif structure and in pesticide and drug development through gene transcription regulation by i-motif structure.

Effect of G-quadruplex loop mutations on the G-quadruplex formation, protein binding and transcription of BmPOUM2 in Bombyx mori.

A G-quadruplex (G4) was identified in the promoter of transcription factor BmPOUM2 in Bombyx mori. This G4 structure contains three loops and is bound by transcription factor BmLARK, facilitating the transcription of BmPOUM2. However, the relationship between the structure and function of the BmPOUM2 G4 remains to be clarified. In this study, loop mutants of the BmPOUM2 G4 structure were generated to study the function of the structure in transcription regulation. The results revealed that mutations of Loops A and B could not completely suppress G4 formation, but affected the binding of the G4 structure with BmLARK and the promoter activity. The mutation (C-to-T) of the one-nucleotide-loop, Loop C, enhanced the G4 formation, its binding with BmLARK and the transcription activity of the BmPOUM2 promoter. It is speculated that the binding site of BmLARK probably is on the G-quartet planes, rather than on the loops, which may assist the maintenance and modification of the G4 structure and its protein binding activity.

Lacking of sex-lethal gene lowers the fertility of male reproduction in Spodoptera litura (Lepidoptera).

Sex-lethal (Sxl) encodes an RNA-binding protein that acts as the switch of sex determination in Drosophila and influences the genitalia formation and gonadal development. However, its sex-determination roles are not conserved in all insects and its role in the gonadal development of Lepidoptera is not well documented. In this study, three splicing variants of Sxl mRNA were identified in Spodoptera litura and they highly expressed in gonads, particularly in the testis. The mRNA levels of SlSxl exhibited higher expression in the spermatid than the testis sheaths, and gradually increased with the spermiogenesis. Sex-lethal protein (SlSXL) is mainly distributed in the cytoplasm of spermatocytes and the head of spermatid. Knockout of SlSxl resulted in fewer eupyrene sperm bundles and apyrene sperm bundles in the testes of moth and a large number of undeveloped spermatocysts retained in the moth of mutant testis, and leading to the reduction of oviposition and hatch rate in the offsprings after mating with female. These results suggest that SlSxl is a critical player in the spermiogenesis of S. litura.

20E-mediated regulation of BmKr-h1 by BmKRP promotes oocyte maturation.

BACKGROUND: Krüppel homolog 1 (Kr-h1) is a critical transcription factor for juvenile hormone (JH) signaling, known to play a key role in regulating metamorphosis and adult reproduction in insects. Kr-h1 can also be induced by molting hormone 20-hydroxyecdysone (20E), however, the underlying mechanism of 20E-induced Kr-h1 expression remains unclear. In the present study, we investigated the molecular mechanism of Kr-h1 induction by 20E in the reproductive system of a model lepidopteran insect, Bombyx mori. RESULTS: Developmental and tissue-specific expression analysis revealed that BmKr-h1 was highly expressed in ovaries during the late pupal and adult stages and the expression was induced by 20E. RNA interference (RNAi)-mediated depletion of BmKr-h1 in female pupae severely repressed the transcription of vitellogenin receptor (VgR), resulting in the reduction in vitellogenin (Vg) deposition in oocytes. BmKr-h1 specifically bound the Kr-h1 binding site (KBS) between - 2818 and - 2805 nt upstream of BmVgR and enhanced the transcription of BmVgR. A 20E cis-regulatory element (CRE) was identified in the promoter of BmKr-h1 and functionally verified using luciferase reporter assay, EMSA and DNA-ChIP. Using pull-down assays, we identified a novel transcription factor B. mori Kr-h1 regulatory protein (BmKRP) that specifically bound the BmKr-h1 CRE and activated its transcription. CRISPR/Cas9-mediated knockout of BmKRP in female pupae suppressed the transcription of BmKr-h1 and BmVgR, resulting in arrested oogenesis. CONCLUSION: We identified BmKRP as a new transcription factor mediating 20E regulation of B. mori oogenesis. Our data suggests that induction of BmKRP by 20E regulates BmKr-h1 expression, which in turn induces BmVgR expression to facilitate Vg uptake and oogenesis.

G-Quadruplex Regulation of VEGFA mRNA Translation by RBM4.

In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5'-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5'-UTR of vascular endothelial growth factor A (VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.

The TGF-ß Receptor Gene Saxophone Influences Larval-Pupal-Adult Development in Tribolium castaneum.

The transforming growth factor-ß (TGF-ß) superfamily encodes a large group of proteins, including TGF-ß isoforms, bone morphogenetic proteins and activins that act through conserved cell-surface receptors and signaling co-receptors. TGF-ß signaling in insects controls physiological events, including growth, development, diapause, caste determination and metamorphosis. In this study, we used the red flour beetle, Tribolium castaneum, as a model species to investigate the role of the type I TGF-ß receptor, saxophone (Sax), in mediating development. Developmental and tissue-specific expression profiles indicated Sax is constitutively expressed during development with lower expression in 19- and 20-day (6th instar) larvae. RNAi knockdown of Sax in 19-day larvae prolonged developmental duration from larvae to pupae and significantly decreased pupation and adult eclosion in a dose-dependent manner. At 50 ng dsSax/larva, Sax knockdown led to an 84.4% pupation rate and 46.3% adult emergence rate. At 100 ng and 200 ng dsSax/larva, pupation was down to 75.6% and 50%, respectively, with 0% adult emergence following treatments with both doses. These phenotypes were similar to those following knockdowns of 20-hydroxyecdysone (20E) receptor genes, ecdysone receptor (EcR) or ultraspiracle protein (USP). Expression of 20E biosynthesis genes disembodied and spookier, 20E receptor genes EcR and USP, and 20E downstream genes BrC and E75, were suppressed after the Sax knockdown. Topical application of 20E on larvae treated with dsSax partially rescued the dsSax-driven defects. We can infer that the TGF-ß receptor gene Sax influences larval-pupal-adult development via 20E signaling in T. castaneum.

Identification of LARK as a novel and conserved G-quadruplex binding protein in invertebrates and vertebrates.

Double-stranded DNAs are usually present in the form of linear B-form double-helix with the base pairs of adenine (A) and thymine (T) or cytosine (C) and guanine (G), but G-rich DNA can form four-stranded G-quadruplex (G4) structures, which plays important roles in transcription, replication, translation and protection of telomeres. In this study, a RNA recognition motif (RRM)-containing protein, BmLARK, was identified and demonstrated to bind G4 structures in the promoters of a transcription factor BmPOUM2 and other three unidentified genes of Bombyx mori, as well as three well-defined G4 structures in the human genes. Homologous LARKs from Bombyx mori, Drosophila melanogaster, Mus musculus and Homo sapiens bound G4 structures in BmPOUM2 and other genes in B. mori and H. sapiens. Upon binding, LARK facilitated the formation and stability of the G4 structure, enhancing the transcription of target genes. The G4 structure was visualized in vivo in cells and testis from invertebrate B. mori and vertebrate Chinese hamster ovary (CHO) cells. The results of this study strongly suggest that LARK is a novel and conserved G4-binding protein and that the G4 structure may have developed into an elaborate epigenetic mechanism of gene transcription regulation during evolution.

Antagonistic actions of juvenile hormone and 20-hydroxyecdysone within the ring gland determine developmental transitions in Drosophila.

In both vertebrates and insects, developmental transition from the juvenile stage to adulthood is regulated by steroid hormones. In insects, the steroid hormone, 20-hydroxyecdysone (20E), elicits metamorphosis, thus promoting this transition, while the sesquiterpenoid juvenile hormone (JH) antagonizes 20E signaling to prevent precocious metamorphosis during the larval stages. However, not much is known about the mechanisms involved in cross-talk between these two hormones. In this study, we discovered that in the ring gland (RG) of Drosophila larvae, JH and 20E control each other's biosynthesis. JH induces expression of a Krüppel-like transcription factor gene Kr-h1 in the prothoracic gland (PG), a portion of the RG that produces the 20E precursor ecdysone. By reducing both steroidogenesis autoregulation and PG size, high levels of Kr-h1 in the PG inhibit ecdysteriod biosynthesis, thus maintaining juvenile status. JH biosynthesis is prevented by 20E in the corpus allatum, the other portion of the RG that produces JH, to ensure the occurrence of metamorphosis. Hence, antagonistic actions of JH and 20E within the RG determine developmental transitions in Drosophila Our study proposes a mechanism of cross-talk between the two major hormones in the regulation of insect metamorphosis.

Bombyx mori transcription factors FoxA and SAGE divergently regulate the expression of wing cuticle protein gene 4 during metamorphosis.

Stage-specific gene expression governs metamorphosis of the silkworm, Bombyx mori. B. mori wing cuticle protein gene 4 (BmWCP4) is an essential gene for wing disc development expressed specifically during pupation. BmWCP4 transcription is suppressed at the larval stage by unknown mechanisms, which we sought to elucidate here. Bioinformatics analysis predicted seven potential Forkhead box (Fox) cis-regulatory elements (CREs) in the BmWCP4 promoter region, and we found that Fox CRE6 contributes to suppression of BmWCP4 expression. Electrophoretic mobility shift (EMSA) and DNA pull-down assays revealed that BmFoxA suppressed activity at the BmWCP4 promoter by specifically binding to the Fox CRE6. The expression level of BmFoxA in the wing discs was higher during the larval stage than at the pupal stage. In contrast, expression of another transcription factor, BmSAGE, increased over the course of development. Of note, the hormone 20-hydroxyecdysone (20E), which governs molting in insects, suppressed BmFoxA expression in the wing discs and up-regulated that of BmSage EMSA and cell co-transfection assays indicated that BmSAGE interacted with BmFoxA and suppressed its binding to the Fox CRE6, thereby releasing BmFoxA-mediated suppression of BmWCP4 In summary, higher BmFoxA expression during the larval stage suppresses BmWCP4 expression by binding to the Fox CRE6 on the BmWCP4 promoter. During metamorphosis, BmSAGE forms a complex with BmFoxA to relieve this repression, initiating BmWCP4 expression. Taken together, this study reveals a switchlike role for BmFoxA in regulating BmWCP4 expression and provides new insights into the regulatory regulation of wing disc development in insects.

Transcriptomic analysis of the testicular fusion in Spodoptera litura.

BACKGROUND: Lepidoptera is one group of the largest plant-feeding insects and Spodoptera litura (Lepidoptera: Noctuidae) is one of the most serious agricultural pests in Asia countries. An interesting and unique phenomenon for gonad development of Lepidoptera is the testicular fusion. Two separated testes fused into a single one during the larva-to-pupa metamorphosis, which is believed to contribute to sperm production and the prevalence in field. To study the molecular mechanism of the testicular fusion, RNA sequencing (RNA-seq) experiments of the testes from 4-day-old sixth instar larvae (L6D4) (before fusion), 6-day-old sixth instar larvae (L6D6, prepupae) (on fusing) and 4-day-old pupae (P4D) (after fusion) of S. litura were performed. RESULTS: RNA-seq data of the testes showed that totally 12,339 transcripts were expressed at L6D4, L6D6 and P4D stages. A large number of differentially expressed genes (DEGs) were up-regulated from L6D4 to L6D6, and then more genes were down-regulated from L6D6 to P4D. The DEGs mainly belongs to the genes related to the 20E signal transduction pathway, transcription factors, chitin metabolism related enzymes, the families of cytoskeleton proteins, extracellular matrix (ECM) components, ECM-related protein, its receptor integrins and ECM-remodeling enzymes. The expression levels of these genes that were up-regulated significantly during the testicular fusion were verified by qRT-PCR. The matrix metalloproteinases (MMPs) were found to be the main enzymes related to the ECM degradation and contribute to the testicular fusion. The testis was not able to fuse if MMPs inhibitor GM6001 was injected into the 5th abdomen region at L6D6 early stage. CONCLUSIONS: The transcriptome and DEGs analysis of the testes at L6D4, L6D6 and P4D stages provided genes expression information related to the testicular fusion in S. litura. These results indicated that cytoskeleton proteins, ECM-integrin interaction genes and ECM-related proteins were involved in cell migration, adhesion and fusion during the testicular fusion. The ECM degradation enzymes MMPs probably play a critical role in the fusion of testis.

BmILF and i-motif structure are involved in transcriptional regulation of BmPOUM2 in Bombyx mori.

Guanine-rich and cytosine-rich DNA can form four-stranded DNA secondary structures called G-quadruplex (G4) and i-motif, respectively. These structures widely exist in genomes and play important roles in transcription, replication, translation and protection of telomeres. In this study, G4 and i-motif structures were identified in the promoter of the transcription factor gene BmPOUM2, which regulates the expression of the wing disc cuticle protein gene (BmWCP4) during metamorphosis. Disruption of the i-motif structure by base mutation, anti-sense oligonucleotides (ASOs) or inhibitory ligands resulted in significant decrease in the activity of the BmPOUM2 promoter. A novel i-motif binding protein (BmILF) was identified by pull-down experiment. BmILF specifically bound to the i-motif and activated the transcription of BmPOUM2. The promoter activity of BmPOUM2 was enhanced when BmILF was over-expressed and decreased when BmILF was knocked-down by RNA interference. This study for the first time demonstrated that BmILF and the i-motif structure participated in the regulation of gene transcription in insect metamorphosis and provides new insights into the molecular mechanism of the secondary structures in epigenetic regulation of gene transcription.

Genome-wide patterns of copy number variations in Spodoptera litura.

Spodoptera litura is a polyphagous pest and can feed on more than 100 species of plants, causing great damage to agricultural production. The SNP results showed that there were gene exchanges between different regions. To explore the variations of larger segments in S. litura genome, we used genome resequencing samples from 14 regions of China, India, and Japan to study the copy number variations (CNVs). We identified 3976 CNV events and 1581 unique copy number variation regions (CNVRs) occupying the 108.5 Mb genome of S. litura. A total of 5527 genes that overlapped with CNVRs were detected. Selection signal analysis identified 19 shared CNVRs and 105 group-specific CNVRs, whose related genes were involved in various biological processes in S. litura. We constructed the first CNVs map in S. litura genome, and our findings will be valuable for understanding the genomic variations and population differences of S. litura.

Ras-Raf-MAPK signaling promotes nuclear localization of FOXA transcription factor SGF1 via Ser91 phosphorylation.

Ras-Raf-MAPK signaling promotes cell proliferation and cell survival. We previously reported that Ras1CA overexpression, specifically in the posterior silk glands (PSGs) of the silkworm Bombyx mori, increased fibroin synthesis and cell size, resulting in improved silk yields. In this study, we compared the iTRAQ-based phosphoproteomic profiles of PSGs from wild-type and Ras1CA-overexpressing silkworms. Silk gland factor 1 (SGF1), a FOXA transcription factor that plays a critical role in activating fibroin gene expression, was identified as a phosphoprotein harboring Ser91 as a potential MAPK phosphorylation site. Ser91 phosphorylation of SGF1 was enhanced by Ras1CA overexpression, and this finding was verified by selected reaction monitoring. Consistently, MAPK activity is well correlated with Ser91 phosphorylation of SGF1 and its nuclear localization in PSG cells during silkworm development. Ras1CA overexpression and treatment with inhibitors of Ras signaling promoted or inhibited SGF1 nuclear localization, respectively; mutation of Ser91 to Ala91 eliminated SGF1 nuclear localization. Moreover, MAPK binds to SGF1 and directly phosphorylates Ser91, demonstrating Ser91 as a MAPK phosphorylation site in SGF1. In conclusion, Ras-Raf-MAPK signaling promotes SGF1 nuclear localization for transactivation via Ser91 phosphorylation in silkworms, showing that FOXA transcription factors are regulated via MAPK phosphorylation in animals.

Fat Body Biology in the Last Decade.

The insect fat body is analogous to vertebrate adipose tissue and liver. In this review, the new and exciting advancements made in fat body biology in the last decade are summarized. Controlled by hormonal and nutritional signals, insect fat body cells undergo mitosis during embryogenesis, endoreplication during the larval stages, and remodeling during metamorphosis and regulate reproduction in adults. Fat body tissues are major sites for nutrient storage, energy metabolism, innate immunity, and detoxification. Recent studies have revealed that the fat body plays a central role in the integration of hormonal and nutritional signals to regulate larval growth, body size, circadian clock, pupal diapause, longevity, feeding behavior, and courtship behavior, partially by releasing fat body signals to remotely control the brain. In addition, the fat body has emerged as a fascinating model for studying metabolic disorders and immune diseases. Potential future directions for fat body biology are also proposed herein.

New insight into the mechanism underlying the silk gland biological process by knocking out fibroin heavy chain in the silkworm.

BACKGROUND: Exploring whether and how mutation of silk protein contributes to subsequent re-allocation of nitrogen, and impacts on the timing of silk gland degradation, is important to understand silk gland biology. Rapid development and wide application of genome editing approach in the silkworm provide us an opportunity to address these issues. RESULTS: Using CRISPR/Cas9 system, we successfully performed genome editing of Bmfib-H. The loss-of-function mutations caused naked pupa and thin cocoon mutant phenotypes. Compared with the wild type, the posterior silk gland of mutant showed obviously degraded into fragments in advance of programmed cell death of silk gland cells. Comparative transcriptomic analyses of silk gland at the fourth day of the fifth instar larval stage(L5D4)identified 1456 differential expressed genes (DEGs) between posterior silk gland (PSG) and mid silk gland (MSG) and 1388 DEGs between the mutant and the wild type. Hierarchical clustering of all the DEGs indicated a remarkable down-regulated and an up-regulated gene clade in the mutant silk glands, respectively. Down-regulated genes were overrepresented in the pathways involved in cancer, DNA replication and cell proliferation. Intriguingly, up-regulated DEGs are significantly enriched in the proteasome. By further comparison on the transcriptome of MSG and PSG between the wild type and the mutant, we consistently observed that up-regulated DEGs in the mutant PSG were enriched in protein degrading activity and proteasome. Meantime, we observed a series of up-regulated genes involved in autophagy. Since these protein degradation processes would be normally occur after the spinning time, the results suggesting that these progresses were activated remarkably ahead of schedule in the mutant. CONCLUSIONS: Accumulation of abnormal fib-H protein might arouse the activation of proteasomes as well as autophagy process, to promote the rapid degradation of such abnormal proteins and the silk gland cells. Our study therefore proposes a subsequent process of protein and partial cellular degradation caused by mutation of silk protein, which might be helpful for understanding its impact of the silk gland biological process, and further exploration the re-allocation of nitrogen in the silkworm.

A regulatory pathway, ecdysone-transcription factor relish-cathepsin L, is involved in insect fat body dissociation.

Insect fat body is the organ for intermediary metabolism, comparable to vertebrate liver and adipose tissue. Larval fat body is disintegrated to individual fat body cells and then adult fat body is remodeled at the pupal stage. However, little is known about the dissociation mechanism. We find that the moth Helicoverpa armigera cathepsin L (Har-CL) is expressed heavily in the fat body and is released from fat body cells into the extracellular matrix. The inhibitor and RNAi experiments demonstrate that Har-CL functions in the fat body dissociation in H. armigera. Further, a nuclear protein is identified to be transcription factor Har-Relish, which was found in insect immune response and specifically binds to the promoter of Har-CL gene to regulate its activity. Har-Relish also responds to the steroid hormone ecdysone. Thus, the dissociation of the larval fat body is involved in the hormone (ecdysone)-transcription factor (Relish)-target gene (cathepsin L) regulatory pathway.